Directed evolution

I) Kemp eliminase

 * 2rkx
 * 3iio
 * 3iip
 * 3iiv



A series of computationally designed enzymes that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has TIM barrel scaffold. The Kemp elimination of 5-nitrobenzisoxazole was chosen as a model reaction for proton (H) transfer from carbon, simultaneously with the cut of the nitrogen–oxygen (N-O) bond, resulting in cyanophenol product. Such reaction is a critical step in many enzymatic reactions. The catalytic base (E101), the general acid/H-bond donor (K222), and the stacking residue (W50) make interactions with the 5-nitrobenzisoxazole at the active site of KE07. Directed evolution can significantly improve the stability, expression and activity of enzymes. In the catalytically improved directed evolutionary variants of KE07 containing the Ile7Asp mutation, Asp7 breaks the Glu101–Lys222 salt bridge (for example 3iiv, chain A is shown).

The comparison of the designed structure (colored orange) modelled in the presence of the 5-nitrobenzisoxazole and unbound crystal structure (2rkx, colored lime) of KE07 shows only limited conformational changes. In the designed structure, the amino group of Lys222 is ~4 Å away from the transition state phenolic oxygen, to stabilize the negative charge of the product phenoxide. However, Lys222 can also form a weak salt bridge with the catalytic Glu101 with a distance of ~3.6 Å. In the crystal structure of the KE07 without ligand (2rkx), the Glu101–Lys222 distance is 2.84 Å, i.g. within salt bridge distance. This is probably a unique feature of the unbound conformation of KE07. As was mentioned above, in the catalytically improved directed evolutionary mutants of KE07 bearing the Ile7Asp mutation, Asp7 breaks the Glu101–Lys222 salt bridge (in the evolved mutants, the NεLys222–OγGlu101 distance is 3.3–5.7 Å), in some cases directly (as in the present case 3iiv, chain A) interacting with Lys222 (the NεLys222–OβAsp7 distance is 2.8–5.7 Å). An overlap of the structures of the wildtype (lime) KE07 and the its <font color='lightskyblue'>evolved Ile7Asp mutant reveals how the Ile7Asp mutation causes the shift of the Lys222 side chain away from Glu101.

The residues Arg5, Glu46, Lys99, and Glu167 of <font color='lime'>KE07 unbound wildtype crystal structure (2rkx) form <scene name='3iio/Ali1/1'>electrostatic network at the bottom of the active site. In this case Lys222, of course, is not involved in this network, because it could not form electrostatic interaction with Ile7. Ile7Asp mutation in the evolved mutants introduces Lys222 to this electrostatic network or <scene name='3iio/Ali1/3'>directly as <font color='lightskyblue'>in case of 3iiv<font color='lightskyblue'>, chain A, or <scene name='3iio/Ali1/4'>via water molecule (3iiv, <font color='tan'>chain B ).

The crystal structures of the catalytically improved directed evolutionary KE07 mutants also demonstrate that replacement of side chains via mutations, combined with minor backbone changes, could allowed the new enzyme–substrate interactions. For example, <scene name='3iio/Ali1/5'>superposition of the structures of the <font color='orange'>KE07 design and evolved <font color='darkmagenta'>KE07 round 4 1E/11H chain A (3iio) reveals that the mutation Gly202Arg caused a shift of the adjacent loop (residues 175–177) and could introduce a new interaction with the nitro group of the 5-nitrobenzisoxazole. The directed evolution also creates new interaction networks of charged surface residues at the upper part of the active site. In the <scene name='3iio/Ali1/6'>wildtype KE07 (2rkx, <font color='lime'>colored lime ), Gly is in the position 202, Asn is in the position 224, and distance between Asn224 O and His201 N is 7.9 Å. In the evolved variants, following the Gly202Arg and Asn224Asp mutations, Asp224 and His201 gradually became closer, with distances between Asn224 O and His201 N of 4.6 Å in the <scene name='3iio/Ali1/7'>round 4 variant (3iio, <font color='darkmagenta'>colored darkmagenta ) and 3.6 Å in the <scene name='3iio/Ali1/8'>round 7 variant, chain B (3iiv, <font color='tan'>colored tan ). In rounds 6-7 variants, Asp224 can potentially interact with Arg202 and with His201. This network of Arg202–Asp224–His201 also brings His201 closer to the substrate (not shown). Interestingly, the <scene name='3iio/Ali1/9'>conformation of Trp50 at the active site in <font color='lightskyblue'>chain A of round 7 1/3H variant (3iiv) significantly differs from those in all other structures, including <font color='tan'>chain B within the asymmetric unit of round 7 1/3H. Of note, that Trp50 of chain A overlaps the substrate.

</StructureSection>

II) Colicin7 and Immunity proteins

 * 3gkl
 * 3gjn

<StructureSection load='3gkl' size='500' frame='true' align='right' scene='3gkl/Al/1' > Iterative rounds of random mutagenesis and selection of immunity protein 9 (colored yellow) toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation <scene name='3gkl/Al/3'>variants <font color='lime'>R12-2 (3gkl; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and <font color='darkred'>R12-13 (3gjn; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved.

<scene name='3gkl/Align/2'>Structural alignment of the immunity protein 9 (Im9, 1bxi, colored yellow), <font color='lime'>evolved variant R12-2 (lime), and <font color='blue'>immunity protein 7 (Im7, 7cei, colored blue) reveals their structural identity. However, when the immunity proteins-bound <scene name='3gkl/Align/3'>colicins within their complexes were aligned, they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 (1bxi) there are Tyr54 and Tyr55, while in the Im7 (7cei) Tyr55 and Tyr56 are homologous to them. The most <scene name='3gkl/Align/4'>prominent differences are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and <font color='lime'>evolved variant R12-2 (lime, labeled in black). This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28.

Comparison of the different Im-colicin complexes reveals changes in the binding configuration of the evolved variants which increase affinity toward ColE7 by re-aligning pre-existing Im9 residues. Glu30 of Im9 (1bxi, colored yellow) forms <scene name='3gkl/Active_site/3'>double salt bridge with Arg54 of <font color='orange'>ColE9 (orange), whereas Asp51 have not direct side chain–side chain interactions. <font color='blue'>Asp31 of Im7 (blue) (corresponding to Im9 Glu30) is involved in <scene name='3gkl/Active_site/6'>cluster of salt bridge bonds to <font color='darkmagenta'>Arg520 and Lys525 of ColE7 (darkmagenta), while <font color='blue'>Asp52 of Im7 (corresponding to Im9 Asp51) is within hydrogen bond distance to <font color='darkmagenta'>Thr531 and Arg530 of ColE7. <font color='lime'>Glu30 in the variant R12-2 (lime) is shifted and forms a <scene name='3gkl/Active_site/8'>double salt bridge to <font color='magenta'>Arg520 of ColE7 (magenta). <font color='lime'>Asp51 is within hydrogen bond distance to <font color='magenta'>Thr531 of ColE7. However, the side chains of <font color='magenta'>Lys525 and Arg530, which are very important in salt bridge contacts with Glu30 and Asp51, respectively, in the structure of the ColE7–Im7 complex have a different conformation that eliminates these contacts in evolved variant R12-2.

In the <scene name='3gkl/Mut/2'>Im9 <font color='magenta'>Val37 (colored magenta) forms stabilizing hydrogen bond with Leu33. In the <scene name='3gkl/Mut/3'>evolved variant R12-2, <font color='darkmagenta'>Ile37 (colored darkmagenta) interacts with two additional residues, Tyr54 and Ser50. Moreover, <font color='darkmagenta'>Ile37 also forms additional hydrogen bond with Gly41 and can thereby have enabled the appearance of the selectivity mutation E41G.

In contrast to the <font color='lime'>evolved variant R12-2 (3gkl), the <font color='cyan'>evolved variant R12-13 (3gjn) carries the <scene name='3gkl/Align/10'>Tyr55Trp mutation in the conserved region. Both <font color='lime'>Tyr55 in R12-2 and <font color='cyan'>Trp55 in R12-13 could sustain the hydrophobic core and create a <scene name='3gkl/Align/11'>hydrogen bond to Lys528 backbone (3gkl colicin residues are colored in <font color='magenta'>magenta, 3gjn colicin residues are colored <font color='blueviolet'>blueviolet ). However, the additional bulkiness of the Trp contributes in expanding its <scene name='3gkl/Align/9'>hydrophobic interactions to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530.

The <scene name='3gkl/Ali/1'>overall conformation of the two evolved variants <font color='lime'>R12-2 (3gkl) and <font color='cyan'>R12-13 (3gjn) is very similar. The variant <font color='lime'>R12-2 carries <scene name='3gkl/Ali/2'>mutation E41G. In the bound wildtype Im9 (yellow) Glu41 makes a <scene name='3gkl/Ali/3'>salt bridge with the <font color='orange'>ColE9’s Lys97 (1bxi). While in the <font color='blueviolet'>R12-13 /<font color='cyan'>ColE7 complex the <font color='blueviolet'>closest ColE7 residues <scene name='3gkl/Ali/4'>contacting <font color='cyan'>R12-13 Glu41 are <font color='blueviolet'>Thr531 (3.37Å) and Lys528 (8.85Å) (3gjn). In the <font color='lime'>R12-2 /<font color='magenta'>ColE7 complex the <scene name='3gkl/Ali/5'>closest <font color='magenta'>ColE7 residue to <font color='lime'>R12-2 Gly41 is <font color='magenta'>Thr531 (9.48Å) (3gkl).

</StructureSection>